Abstract

Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.

Single vesicle spectral unmixing of astrocytes after AO loading. Examples of normalized emission spectra of astroglial vesicular organelles, after loading with 5 μM AO, 15 min at 23°C (A), and at 37°C (B), respectively. Although extracted from an Airy-sized region of interest centered on the organelle, these spectra may contain signals from the organelles from above and below focus. To unambiguously detect the presence of monomeric (m.AO) or dimeric AO (d.AO), we used a statistical test that compares the organelle spectrum with its (peripheral) background. Green and red color-code for organelles on which m.AO or d.AO only were detected; yellow indicates the detection of both, i.e., colocalization on the same organelle. (C) Detection of m.AO and d.AO in single organelles. Each barbell represents one organelle. Their extremities give the result of the detection test for the presence of m.AO and d.AO, respectively. The dashed line indicates the threshold level corresponding to 95% certainty for detecting fluorophore presence in the studied spot. Green barbells symbolize organelles that exclusively contain m.AO; red, only d.AO; and yellow stands for organelles in which both the presence of m.AO and d.AO was detected (see ). Error bars are confidence intervals defined for 70% certainty.

AO obscures the EGFP signal on spectral images. (A) Examples of normalized mixed organelle spectra extracted from a VAMP2-EGFP expressing astrocyte before (top) and after (bottom) AO labeling (5 μM, 15 min, 23°). Note the variable degree of red hue. Reconstituted fluorophore abundance maps (left, and confidence interval of the SILU estimate, right, see Materials and Methods) before (B) and after AO loading (C). EGFP, but not acridine orange monomers (m.AO) or dimers (d.AO), were detected before AO labeling. After AO accumulation in the vesicle lumen, the EGFP signal is totally obscured by the bright m.AO and d.AO fluorescence. Units are numbers of pure spectra of EGFP, m.AO, and d.AO, respectively. Exposure times were 100 ms before and 10 ms after AO loading, respectively. Scale bar is 1 μm. The hole in the postloading EGFP image is due to the absence of a nonnegativity constraint on the abundance coefficients. (D) Log-intensity histogram of green fluorescence (TIRF 488 nm, HQ535/50m) of near-membrane organelles expressing VAMP2-EGFP (open bars), or labeled with AO during 15 min, with 5 μM [AO] at 23°C (gray) or 37°C (red). Graph pools data from 35 cells.